| Summary: | 碩士 === 國立臺灣大學 === 畜產學研究所 === 90 === 英 文 摘 要
The feasibility of using spermatozoa as a gene vector
for generation of transgenic chicken
Cho-Chen Yang
Abstract
The aim of this present study was to establish an available system that is characterized by convenience, high reproducibility and transgenic efficiency for generation of transgenic (Tg) chicken. To meet these purposes, three experiments were conducted, by using CX-EGFP (3.3 Kb) and/or ORF-EGFP (2.9 Kb) as the reporter gene(s); and the feasibility of using spermatozoa as a gene vector for carrying transgene(s) into chicken ovulated eggs were examined against to two different strategies, including the sperm-mediated gene transfer (SMGT) and the testis-mediated gene transfer (TMGT). Of the SMGT strategy, ejaculated cock spermatozoa that had been previously treated in vitro with the mixture(s) of either liposome/transgene or liposome-like/transgene for allowing them to associate with liposome/transgene and liposome-like/transgene complex, respectively, were subjected to molecular analyses of transgene(s) existed within spermatozoa and/or using for artificial insemination (AI) to the laying hens. Of the TMGT strategy, however, intra-testicular injections of a mixture containing liposome-like/transgene complex had been surgically performed against to each cock by 10 ~ 22 days before they were subjected to semen collection for molecular analyses of transgene(s) existed within spermatozoa and/or using for AI to those laying hens.
Results obtained from the initial experiment appeared that chicken spermatozoa do possess capability to take up the foreign DNA; this was confirmed by evidences of PCR and Southern-hybridization analyses, both showing positive signals of transgene(s) existed within sperm when chicken ejaculated spermatozoa had been co-cultured at 18 ℃ with CX-EGFP/liposome complexes for 30 min. and subsequently treated with DNase I for 1 hr in vitro.
In the second experiment, cock spermatozoa that had been pre-treated in vitro with CX-EGFP/liposome and/or CX-EGFP/liposome—like complex were further used for AI to each of 6 laying hens and eggs oviposited between day-3 and day-7 post AI were collected and subjected to incubation. Of these study, two out of 53 from CX-EGFP/liposome treated group and two out of 21 from CX-EGFP/liposome—like treated group of the newly hatched chicken were respectively confirmed to be transgenic, both based on evidences of PCR and Southern-hybridization analyses.
In the final studies, the in vivo effectiveness of liposome-like enduing the testicular germ cells with capability to integrate the foreign DNA were confirmed by both PCR- and Southern-hybridization-based results against to the genomic DNA (gDNA) extracted from cock ejaculated spermatozoa obtained at day-10, day-19, and day-22 after they had accepted the intra-testicular injection of a mixture containing CX-EGFP/liposome-like and ORF-EGFP/liposome-like complexes. Moreover, two out of four and two out of ten of the newly hatched chicken generated from laying hens that had been AI with the TMGT spermatozoa were confirmed, respectively, to harbor the transgene of CX-EGFP and/or ORF-EGFP in their genome.
According to results of PCR and Southern analyses against to gDNA samples of new born chickens produced from laying hens AI with semen sample(s) that had been pre-treated by DNA / liposome and/or DNA / liposome-like complexes, the conclusion comes to this present study is that both sperm-mediate-based and testis-mediate-based strategies are well feasible for using to generate the Tg chicken.
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